Many strains of microalgae are efficient producers of triacylglycerols, which can be converted for use as biodiesel (Sheehan et al., National Renewable Energy Laboratory, 328, 1998). Algae have been identified as a viable feedstock for biofuels due to their efficient abilities to convert sunlight and CO2 to biomass, synthesize large quantities of lipids (20-75% dry mass), thrive in saline water, grow on non-arable land, and grow in open or closed systems (Sheehan, J et al., National Renewable Energy Laboratory, 328, 1998; Huang, G et al., Applied Energy, 1-9, 2009; Rodolfi, L et al., Biotechnol. Bioeng. 102, 100-112, 2009; Hu, Q et al. Plant J 54, 621-639, 2008; and Spolaore, P. et al., J. Biosci. Bioeng. 101, 87-96, 2006). Microalgae are considered to be superior oil-producers compared to terrestrial competitors (e.g. corn, palm, rapeseed, jatropha, and soybean) because microalgae devote fewer resources to the synthesis of structural components such as cellulose and lignin (Song D. et al., Chin J Biotechnol 24, 341-348, 2008; Chisti, Y et al., Biotechnol. Adv. 25, 294-306, 2007; and Chisti, Y et al. Trends Biotechnol. 26, 126-131, 2008). While nitrogen-deficient conditions lead to an increase in lipid/cell, there is an overall decrease in the growth and cell-mass produced. Due to the commercial applications of these algae, there is a need to develop a better understanding of their metabolic pathways. Furthermore, a need exists for the development of novel ways to increase triacylglycerol production from algae that are economically competitive and sustainable. One solution for achieving this is to understand and control lipid-producing pathways.
Techniques for increasing lipid production in microalgae include nutrient-limitation and genetic engineering techniques for increasing lipid production in microalgae (James, G O et al., Bioresour. Technol. 102, 3343-3351, 2011; Wang, Z T et al., Eukaryotic Cell 8, 1856-1868, 2009; Lamers, P P et al., Biotechnol. Bioeng. doi:10.1002/bit.22725, 2010; Richardson, B et al., Applied and Environmental Microbiology 18, 245-250, 1969; Suen, Y et al., J Phycol 23, 289-296, 1987; Zhila, N O et al., Botryococcus. J. Appl. Phycol. 17, 309-315, 2005; Siaut, M et al., BMC Biotechnology 11, 7, 2011; and Li, Y et al., Biotechnol. Bioeng. 107, 258-268, 2010). However, these techniques do not allow for real-time, reversible, or temporal control at various stages of cell growth, and require genetic and biochemical knowledge of the pathways involved in lipid production. Moreover, nutrient-limitation also results in lower cell density.
The use of chemical genetics overcomes the problems of nutrient-limitation and genetic engineering techniques by utilizing a phenotypic approach to identify pathways of interest in lipid production that allows for probing of algae biology. Chemical genetics involves the systematic use of small molecules to modify protein function in real-time, rather than genetic mutation methods to disrupt gene function (Schreiber, S, Nat Chem Biol, 2005; Walsh, D P et al., Chem. Rev. 106, 2476-2530, 2006; and Stockwell, B R, Nature, 2004). Additionally, chemical genetics have several advantages over classical genetics, including: 1) real-time control, 2) reversible and temporal control at variable stages of cell growth, 3) access to a “partial knockout” based on concentration effects, 4) overcoming genetic redundancy, and 5) performing sequential treatments of small molecules to accomplish the effect of multiple mutations (Lehár, J et al, Nat Chem Biol 4, 674-681, 2008).
Moreover, while U.S. Patent Application Publication No. US 2011/0078946 disclose the use of compounds that inhibit fatty acid metabolism, such as gluconeogenesis inhibitors, fatty acid oxidation inhibitors, fatty acid transporter inhibitors, reductase inhibitors, isomerase inhibitors, and uncoupling protein inhibitors, to promote the accumulation or storage of fatty acids in plants, algae, or fungi; these compounds only target a single metabolic pathway.
Accordingly there exists a need for the development of a chemical genetics approach for identifying chemical compounds that that increase lipid production, without limiting cell growth or density in algae and yeast, and to increase growth rate in algae and yeast by targeting additional biochemical signaling pathways that control fatty acid and triacylglycerol biosynthesis, storage, and metabolism.